The present invention relates to vaccines, more particularly, to the production of oral vaccines in transgenic edible plants which can produce human papillomavirus (hereinafter, abbreviated xe2x80x98HPVxe2x80x99) like particles.
Papillomavirus, a genus of Papovaviridae, is a double strand DNA tumor virus infecting mammals such as deer, dog, goat, horse, rat and sheep as well as human (Pfister, H., 1987, Adv. Cancer Res., 48, 113-147). The virus infects the basal cells of the epidermis or mucosa of the host to induce papillomas commonly known as warts or verrucas.
About twenty types of the known seventy types of HPV induce benign tumours and involve in development of malignant tumours in the mucosal epithelium of a mouth or a genital tract (Zur Hausen H., 1988, Mol Carcinogenesis 8:147-150). Especially the type of HPV-16 or HPV-18 is the main cause of the cancer of genital tract, particularly of the cervix, which is one of the commonest woman""s cancers, while the HPV-18 is more closely related to the malignancy (Rends M. J., Donaldson Y. K., Duvall E. and Colin C. Bird, 1993, Human Pathology 24: 432-437). In the most cases of Korean women, HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35 are found in the diseased cervix tissues.
According to the World Health Organization, cancers of the cervix have been plagued on five hundred thousand or more women around the world every year and particularly in the less developed countries the cancer of cervix is the main cause of women deaths. In Korea, even though the ratio of the subject suffering from the cancer of cervix to total women has been decreased to 22% from 27.7% of a decade ago, the cancer of cervix is still one of the most spread diseases in women.
The cervical cancer occurs in the anogenital region and is transmitted by sexual contact. Untill now, pap smear, cervicography, papilloma virus detection, colposcopy or in situ hybridization method has been used for diagnosis thereof (Lee, Sang-Sook et al., Korean Met. Association J., 33(1): 89-97). However, since most of said diagnostic methods have shortcomings of displeasure and inaccuracy, a simple diagnostic method such as the method of using an antigen-antibody reaction has been required. Moreover, mass-production or recovery method of using animal cell culture system should be developed for the vaccines to be manufactured.
However, the virions can be produced only in the non-dividing, differentiated keratinocytes and these cells cannot be grown in culture, which has made it difficult to produce the virus particles sufficient to molecular biological studies as well as to develop the preventive or curing vaccines. In addition, since HPV is host-specific, there are also many difficulties in developing the animal model system for testing the efficacy of HPV as a vaccine.
Researches for the vaccine for the cervical cancer are focused on a prophylactic and a therapeutic vaccine. Prophylactic vaccines induce the production of neutralizing antibodies against HPV L1/L2 so that they can prevent the infection of the HPV or further progress of the disease to malignant tumour even when the hosts have been already infected. While the therapeutic vaccines targeting HPV E6/E7 induce a humoral immune response to induce the regression of the lesions or malignant tumour cells.
Up to the present, a part of the HPV capsid proteins produced by recombinant DNA techniques and synthetic peptides have been developed as vaccines for the cancer of cervix. Said recombinant proteins were produced in an established systems such as bacteria, yeasts, baculoviruses and recombinant vaccinia viruses, and they are used as tools for detecting the antibodies in serum, which indicates the ability to induce the humoral or cellular immunity.
Hagensee et al. disclosed a method for producing HPV L1 virus like particles (hereinafter, abbreviated xe2x80x98VLPsxe2x80x99) as live recombinant vaccinia viruses in the culture of mammary cells (Hagensee M. E., Yaegashi N., Galloway D. A., 1993, J. Virol. 67: 315-322). Gao et al. reported that the formation of tumours was inhibited or delayed in the mice immunized with a HPV E6/E7 protein which was produced by said system (Gao L., Chain B., Sinclair C., 1994, J Gen. Viol. 75: 157-164; Meneguzzi G., Cern C., Kieny M. P., 1991, Virology 181: 62-69).
A live recombinant vaccinia virus, however, has a disadvantage in that the probability of random mutation causing the production of replicative compatible viruses is too high like other cases, which requires long-term and expensive clinical trials for practical use. To overcome these problems, viruses whose ability to replicate is defective have been developed but there are no available vaccines yet (Moss B., 1996, Pro. Natl. Acad. Sci. USA, 93: 11341-11348).
Among other researches for vaccines using a bacterial vector, Denis reported that HPV 16 VLPs produced by attenuated Salmonella typhimurium could elicit the production of mucosal or systemic antibodies specific to antigen (Denis, Nardelihaefliger, Richard B. S., Roden, 1997, Infection and immunity 65: 3328-3336). The principle of the use of synthetic peptides as a vaccine is based on the vaccination with only the epitopes necessary for the induction of immune response, e.g., in the HPV 16 E6/E7 an epitope inducing a cytotoxic T lymphocyte-mediated response is disclosed (Ressing M. E., Sette A., Brandt R. M., 1995, J. Immunol. 154: 5934-5943).
However, there are some difficulties in getting the recombinant protein having a proper antigenicity as a vaccine, due to the fact that said protein produced in a prokaryotic expression system such as E. coli does not have a native conformation. It is supported by the report that HPV produced in E. coli cannot stimulate cytotoxic T lymphocytes but the production of the antibodies (Cason J., Khan S. A. and Best J. M., 1994, Vaccine 11: 603-611).
In order to make the protein have much similarity to the conformation of native protein, animal cells can be used as an expression system of the protein but there are still other problems such as susceptibility to contamination, difficulty in purification, and high cost.
In view of aforementioned reasons, vegetables such as tomato and potato transformed with the vectors carrying the gene encoding an antigenic protein are used to produce a virus itself or parts thereof in the native conformation as an antigen, recently. Said transgenic plant itself may be used as an oral vaccine or an edible vaccine.
As an example of recent advances in the above-mentioned field, surface antigen particle of hepatitis B virus (Thalvala Y. F. and C. J. Artzen, 1995, Pro. Natl. Acad Sci. USA 92: 3358-3361) or Norwalk virus like particle (Xi Jiang, Min Wang, David Y. Granham, Mary M. Estes, 1992, J. Virol. 66(11), 6527-6532; Mason H. S., Ball J. M., Artzen C. J., 1996, Pro. Natl. Acad. Sci. USA 93: 5335-5340) was produced in transgenic plants.
Plant transformation for producing viruses has many advantages in cost, safety and availability. Since the infection of HPV takes place in mucosal surface, oral vaccines may be more effective for the induction of a mucosal immunity than parenteral vaccines.
In addition, oral vaccines produced in edible transgenic plants have other advantages in that delivery, storage, and administration thereof are achieved in inexpensive and simple manner as well as the low production cost and high safety. Particularly, the selling price of the edible vaccine may be lowered to such a low level that it can be easily purchased even in less developed countries.
In view of the efforts to produce an oral vaccine for the cancer of cervix, which is inexpensive and easy to be administered, the inventors completed this invention whereby the transgenic plants producing HPV like particles are provided. Also it is ascertained that said VLPs have efficacy in inducing the production of antibodies by the oral administration as well as the intraperitoneal administration.
Recombinant viral antigens, anti-viral vaccines and transgenic plants expressing the same are provided by the present invention.
Also a method for producing said vaccines in transgenic plant and an expression vector used for transforming the plant, which comprises a DNA sequence encoding an HPV capsid protein operably linked to a plant-specific promoter and a gene encoding a selection marker, are provided by the present invention.
More particularly, the present invention provides prophylactic or therapeutic vaccines for the cancer of cervix comprising HPV like particles as a pharmaceutically active ingredient.
In accordance with the present invention, some of disadvantages of the prior arts are overcome by providing antigens produced in edible transgenic plants which antigens are antigenically and physically similar to those currently used in the manufacture of anti-viral vaccines derived from human serum or recombinant yeasts.
Other advantages made by the present invention are convenience of administration, possibility of mass-production, ability to elicit a mucosal immunity as well as a systemic immunity, low cost for production of vaccine and no need for those skilled in vaccination. All of that is attributed to the fact that the antigens are produced by edible plant.
The vaccines of the present invention are provided in the forms of injection solution, orally administrable formulation, spray or patch for preventing or curing the cancer of cervix. Further, diagnostic kits comprising said HPV like particles as antigens plus antibodies thereof are included in the scopes of the present invention.